Direct measurement of the total antioxidant capacity of cereal products

Direct  Measurement  of  the  Total  Antioxidant  Capacity  of  Foods:  The  Quencher  Approach  

Vural  Gökmen,  Arda  Serpen,  Vincenzo  Fogliano    

H A C E T T E P E   UN I V E R S I T Y  

5th  Central  European  Congress  on  Food,    19-­‐22  May  2010,  Bra<slava,  Slovak  Republic  

Key  Drivers  in  Food  Business  

•  HEALTHY  FOODS    – High  in  FIBERS    – High  in  ANTIOXIDANTS    – Low  in  SALT    – Low  in  trans  FAT    – Low  in  SUGARS    

The  Fact:  AOX  topic  is  very  hot!  

 •  In  2009;    

– 900  papers  – 20,000  cita<ons  

•  Number  of  records  has  been  increasing  exponen<ally!  

During the course of this meeting, Dozen of new papers will be published.

How  do  we  measure  the  AOX  capacity  of  foods?  

•  There  are  various  analy<cal  methodologies;    – ABTS    – DPPH    – ORAC    –   etc    

We have very popular ones !

Factors  to  be  considered  in  AOX  assay  

•  Proper<es  of    – An<oxidant  compounds  (solvophobicity)    – Food  matrix  (microstructure)    

For  ACCURATE  and  REPRODUCIBLE  analysis  

Antioxidant / Food

AOXs  in  foods  are  diverse!  NATURALLY  OCCURRING  AOX  COMPOUNDS    

o   Phenolic  compounds    §   i.e.  FERULIC  ACID  

o   Carotenoids    §   i.e.  LUTEIN      

o   Ascorbic  acid    o   Tocopherols    o   Sulphur  compounds    

 NEO  FORMED  AOX  COMPOUNDS  DURING  PROCESSING    

o   Maillard  reac<on  products  §  i.e.  MELANOIDINS      

These AOXs have different solubility behaviours!

Different chemical structures

Different  forms  of  AOXs  in  foods  

AOXs  in  foods  (liquid,  solid)  are  present  in  different  forms.  

SOLUBLE INSOLUBLE

Case:  Ferulic  Acid  in  Cereal  Cell  Walls  

Ferulic  acid  mostly  in  bound  form  

Ferulic  acid  bound  to  arabinosyl  chain  in  the  

primary  cell  wall  

Hydrolysis-­‐Extrac<on  

•  Cereals  are  typical  food  matrices  in  which  AOXs  are  mainly  in  insoluble  form.    

•  Current  procedures  apply  ‘hydrolysis’  to  release  insoluble  antioxidants  ,  then  ‘extraction’  prior  to  measurement.    

Residual  AOX  Ac<vity  in  Alkaline  Treated  Material  

0  

5  

10  

15  

20  

Wheat  Bran     Alkali  Treated  Wheat  Bran  

Barley   Alkali  Treated  Barley  

TEAC

,  mmol  Trolox  /  k

g  insoluble  material  

Serpen,  Capuano,  Fogliano,  Gökmen  J.  Agric.  Food  Chem.  2007,  55,  7676–7681  

Limita<ons  of  Hydrolysis-­‐Extrac<on  Procedure  

•  There  is  NOT  a  generic  extrac<on  solvent  that  can  dissolve  all  AOXs  – NOT  Reproducible  in  inter  laboratory  tests    

•  Hydrolysis  may  take  too  long  – Some  AOXs  are  lost  due  to  oxida<on  giving  unreliable  &  inaccurate  values    

– Underes<mates  true  AOX  capacity  (i.e.  cereals)    – Modifies  food  microstructure,  so  the  resul<ng  AOX  capacity  may  NOT  be  biologically  relevant.    

An  open  problem…  

Do  we  really  need  to  solubilize  AOXs?    

No  

Go  Direct  No  Extrac<on  &  No  Hydrolysis  

Proposed  Approach:  Surface  ReacBon  

AOX  (functional  group)  bound  to  a  solid  matrix  (carrier)  

n   Quenched  radicals  

Free  radicals  

IDEA:    We  don’t  have  to  extract  (solubilize/hydrolize)  AOXs  to  let  them  react  with  free  radicals.    Reac<on  takes  place  at  the  solid  surface  (surface  reac<on  phenomenon)  when  a  radical  solu<on  is  mixed  with  solid  food.    

Serpen,  Capuano,  Fogliano,  Gökmen  J.  Agric.  Food  Chem.  2007,  55,  7676–7681  

Quiz:  Name  this  method  !  

•  Quick  •  Easy    •  New  •  Cheap    •  Reproducible  

so called

QuENCheR

GoDIRECT sense more as an assay of AOX capacity

QUENCHER  with  ABTS  ...  

§  Ground  sample  is  mixed  with  ABTS  solu<on.    

§  Vortexed  for  certain  <me.    §  Centrifuged  to  separate  op<cally  clear  supernatant      

§  Measured  by  spectrophotometry.    

The  QUENCHER  procedure  was  successfully  adapted  to  DPPH,  FRAP  as  well  as  to  ORAC.    

Test  on  Cereals  

Remark: Classical EXTRACTION-HYDROLYSIS procedures underestimates AOX capacity of cereals.

0   20   40   60   80   100   120   140  

Wheat  

Oat    

Rye  

Rice  

Barley  

Buckwheat  

Corn    

Direct  QUENCHER  Procedure  

Ethanol  Extr  +  Water  Extr  +  Hydrolysis  

Methanol  Extr  +  Hydrolysis  

Serpen,  Gökmen,  Pellegrini,  Fogliano  J.  Cereal  Sci.  2008,  48,  816–820  

Food  

Dietary  intake  

Discarded  

Bioavailable(?)  

Bioac<vity  in  blood  and  other    <ssues  

Colon    fermenta<on  

Soluble  

Small  intes<ne  

Unsoluble  

Diges<on  protease  esterase  

Soluble   Unsoluble  

Soluble   Unsoluble  

Bioac<vity  inside  the  intes<nal  environment    

Bioavailable(?)  

Bioavailable  (??)  

QUENCHER measure may reflect potential in vivo activity of AOXs.